Anti-inflammatory compounds

ABSTRACT

Compositions comprising isolated ido-BR1 are described for use in therapy or prophylaxis, including the treatment of inflammatory diseases (for example as anti-inflammatory drugs) and to reduce inflammation. Also described are methods for monitoring the quality of a Cucurbitaceae extract, to processes for producing a Cucurbitaceae extract as well as to Cucurbitaceae extracts obtainable by such processes.

RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationSerial No. PCT/GB2012/000768, filed Oct. 10, 2012, the entire contentsof which are incorporated herein by reference. Foreign priority benefitsare claimed under 35 U.S.C. §119(a)-(d) or 35 U.S.C. §365(b) of Britishapplication number 1117490.1, filed Oct. 11, 2011, the entire contentsof which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to certain iminosugar acids for treatinginflammatory disease (for example as anti-inflammatory drugs) and toreduce inflammation. The invention therefore finds application inmethods for the treatment of inflammatory diseases and for reducinginflammation.

The invention also relates to methods for monitoring the quality of aCucurbitaceae extract, to processes for producing a Cucurbitaceaeextract as well as to Cucurbitaceae extracts obtainable by suchprocesses.

BACKGROUND OF THE INVENTION

The cucumber (Cucumis sativus) is a widely cultivated plant in the gourdfamily Cucurbitaceae, which includes squash. Cucumbers originated inIndia and have been cultivated for at least 3000 years in Western Asia,and probably introduced to other parts of Europe by the Romans. Recordsof cucumber cultivation appear in France in the 9th century, England inthe 14th century, and in North America by the mid-16th century.

Cucumbers and cucumber extracts have long been recognized as havinganti-inflammatory properties, and have been used topically for varioustypes of skin problems, including swelling under the eyes and sunburn.Cucumber was very popular in the ancient civilizations of Egypt, Greeceand Rome, where it was used not only as a food but also for its skinhealing properties.

However, the active component(s) have not been reported.

It has now been discovered that the iminosugar acid ido-BR1 occurs inolder cucumber varieties but is absent in certain modern commercialvarieties. It has been shown to be a major component of certain cucumberfruits, certain squashes and gourds and is the only iminosugar acid incucumber.

Iminosugar acids (ISAs) constitute a subclass of the more widelydistributed class of phytochemicals known as iminosugars. Many knownISAs are phytochemicals, present as secondary metabolites in planttissues (where they may play a role in defense). While iminosugars arewidely distributed in plants (Watson et al. (2001) Phytochemistry 56:265-2951), the iminosugar acids are much less widely distributed.

Thus, the discovery that the botanical distribution of the iminosugaracid ido-BR1 correlates with medicinal plants used for the treatment ofinflammatory disease is of great significance.

The synthesis of ido-BR1 has been described in Fleet et al. (1986)Tetrahedron Lett. 27: 3205-3208.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the surprisingdiscovery that the botanical distribution of ido-BR1 correlates withmedicinal plants used for the treatment of inflammatory diseases. Thus,for the first time ido-BR1 has been identified as an important bioactiveprinciple in established anti-inflammatory herbal medicines.

Thus, according to the invention there is provided(2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid (6-epiBR1;2R,3R,4R,5S-Trihydroxypipecolic acid; ido-BR1) for use in therapy orprophylaxis.

Other aspects and preferred embodiments of the invention are defined anddescribed in the claims set out below.

As used herein, the term ido-BR1 is intended to define sensu stricto acompound of the formula:

but is also to be interpreted sensu lato to include pharmaceuticallyacceptable salts, solvates, metabolites, prodrugs, bioisosteres,derivatives and protected forms thereof.

Those skilled in the art will appreciate that various N-substitutedderivatives of ido-BR1 are likely to share its anti-inflammatoryproperties, and so the pharmaceutically acceptable derivativescontemplated specifically include compounds of the formula:

in which R represents optionally substituted C₁₋₁₅ alkyl, C₁₋₁₅ alkenylor C₁₋₁₅ alkynyl.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patents, patent applications and other referencesmentioned herein are hereby incorporated by reference in theirentireties for all purposes as if each individual publication, patent orpatent application were specifically and individually indicated to beincorporated by reference and the content thereof recited in full.

Definitions

Where used herein and unless specifically indicated otherwise, thefollowing terms are intended to have the following meanings in additionto any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular isto be read to include the plural and vice versa. The term “a” or “an”used in relation to an entity is to be read to refer to one or more ofthat entity. As such, the terms “a” (or “an”), “one or more,” and “atleast one” are used interchangeably herein.

As used herein, the term “comprise,” or variations thereof such as“comprises” or “comprising,” are to be read to indicate the inclusion ofany recited integer (e.g. a feature, element, characteristic, property,method/process step or limitation) or group of integers (e.g. features,element, characteristics, properties, method/process steps orlimitations) but not the exclusion of any other integer or group ofintegers. Thus, as used herein the term “comprising” is inclusive oropen-ended and does not exclude additional, unrecited integers ormethod/process steps.

The phrase “consisting essentially of” is used herein to require thespecified integer(s) or steps as well as those which do not materiallyaffect the character or function of the claimed invention.

As used herein, the term “consisting” is used to indicate the presenceof the recited integer (e.g. a feature, element, characteristic,property, method/process step or limitation) or group of integers (e.g.features, element, characteristics, properties, method/process steps orlimitations) alone.

As used herein, the term “disease” is used to define any abnormalcondition that impairs physiological function and is associated withspecific symptoms. The term is used broadly to encompass any disorder,illness, abnormality, pathology, sickness, condition or syndrome inwhich physiological function is impaired irrespective of the nature ofthe aetiology (or indeed whether the aetiological basis for the diseaseis established). It therefore encompasses conditions arising frominfection, trauma, injury, surgery, radiological ablation, poisoning ornutritional deficiencies.

As used herein, the term “treatment” or “treating” refers to anintervention (e.g. the administration of an agent to a subject) whichcures, ameliorates or lessens the symptoms of a disease or removes (orlessens the impact of) its cause(s) (for example, pathologicalvariegated states). In this case, the term is used synonymously with theterm “therapy”.

Additionally, the terms “treatment” or “treating” refers to anintervention (e.g. the administration of an agent to a subject) whichprevents or delays the onset or progression of a disease or reduces (oreradicates) its incidence within a treated population. In this case, theterm treatment is used synonymously with the term “prophylaxis”.

The term “subject” (which is to be read to include “individual”,“animal”, “patient” or “mammal” where context permits) defines anysubject, particularly a mammalian subject, for whom treatment isindicated. Mammalian subjects include, but are not limited to, humans,domestic animals, farm animals, zoo animals, sport animals and petanimals. In preferred embodiments, the subject is a human.

As used herein, an effective amount of a compound or composition definesan amount that can be administered to a subject without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio, but onethat is sufficient to provide the desired effect, e.g. the treatment orprophylaxis manifested by a permanent or temporary improvement in thesubject's condition. The amount will vary from subject to subject,depending on the age and general condition of the individual, mode ofadministration and other factors. Thus, while it is not possible tospecify an exact effective amount, those skilled in the art will be ableto determine an appropriate “effective” amount in any individual caseusing routine experimentation and background general knowledge. Atherapeutic result in this context includes eradication or lessening ofsymptoms, reduced pain or discomfort, prolonged survival, improvedmobility and other markers of clinical improvement. A therapeutic resultneed not be a complete cure.

As used herein, the term “combination”, as applied to two or morecompounds and/or agents (also referred to herein as the components), isintended to define material in which the two or more compounds/agentsare associated. The terms “combined” and “combining” in this context areto be interpreted accordingly.

The association of the two or more compounds/agents in a combination maybe physical or non-physical. Examples of physically associated combinedcompounds/agents include:

-   -   compositions (e.g. unitary formulations) comprising the two or        more compounds/agents in admixture (for example within the same        unit dose);    -   compositions comprising material in which the two or more        compounds/agents are chemically/physicochemically linked (for        example by crosslinking, molecular agglomeration or binding to a        common vehicle moiety);    -   compositions comprising material in which the two or more        compounds/agents are chemically/physicochemically co-packaged        (for example, disposed on or within lipid vesicles, particles        (e.g. micro- or nanoparticles) or emulsion droplets);    -   pharmaceutical kits, pharmaceutical packs or patient packs in        which the two or more compounds/agents are co-packaged or        co-presented (e.g. as part of an array of unit doses);

Examples of non-physically associated combined compounds/agents include:

-   -   material (e.g. a non-unitary formulation) comprising at least        one of the two or more compounds/agents together with        instructions for the extemporaneous association of the at least        one compound/agent to form a physical association of the two or        more compounds/agents;    -   material (e.g. a non-unitary formulation) comprising at least        one of the two or more compounds/agents together with        instructions for combination therapy with the two or more        compounds/agents;    -   material comprising at least one of the two or more        compounds/agents together with instructions for administration        to a patient population in which the other(s) of the two or more        compounds/agents have been (or are being) administered;    -   material comprising at least one of the two or more        compounds/agents in an amount or in a form which is specifically        adapted for use in combination with the other(s) of the two or        more compounds/agents.

As used herein, the term “combination therapy” is intended to definetherapies which comprise the use of a combination of two or morecompounds/agents (as defined above). Thus, references to “combinationtherapy”, “combinations” and the use of compounds/agents “incombination” in this application may refer to compounds/agents that areadministered as part of the same overall treatment regimen. As such, theposology of each of the two or more compounds/agents may differ: eachmay be administered at the same time or at different times. It willtherefore be appreciated that the compounds/agents of the combinationmay be administered sequentially (e.g. before or after) orsimultaneously, either in the same pharmaceutical formulation (i.e.together), or in different pharmaceutical formulations (i.e.separately). Simultaneously in the same formulation is as a unitaryformulation whereas simultaneously in different pharmaceuticalformulations is non-unitary. The posologies of each of the two or morecompounds/agents in a combination therapy may also differ with respectto the route of administration.

As used herein, the term “pharmaceutical kit” defines an array of one ormore unit doses of a pharmaceutical composition together with dosingmeans (e.g. measuring device) and/or delivery means (e.g. inhaler orsyringe), optionally all contained within common outer packaging. Inpharmaceutical kits comprising a combination of two or morecompounds/agents, the individual compounds/agents may unitary ornon-unitary formulations. The unit dose(s) may be contained within ablister pack. The pharmaceutical kit may optionally further compriseinstructions for use.

As used herein, the term “pharmaceutical pack” defines an array of oneor more unit doses of a pharmaceutical composition, optionally containedwithin common outer packaging. In pharmaceutical packs comprising acombination of two or more compounds/agents, the individualcompounds/agents may unitary or non-unitary formulations. The unitdose(s) may be contained within a blister pack. The pharmaceutical packmay optionally further comprise instructions for use.

As used herein, the term “patient pack” defines a package, prescribed toa patient, which contains pharmaceutical compositions for the wholecourse of treatment. Patient packs usually contain one or more blisterpack(s). Patient packs have an advantage over traditional prescriptions,where a pharmacist divides a patient's supply of a pharmaceutical from abulk supply, in that the patient always has access to the package insertcontained in the patient pack, normally missing in patientprescriptions. The inclusion of a package insert has been shown toimprove patient compliance with the physician's instructions.

The combinations of the invention may produce a therapeuticallyefficacious effect relative to the therapeutic effect of the individualcompounds/agents when administered separately.

The term bioisostere (or simply isostere) is a term of art used todefine drug analogues in which one or more atoms (or groups of atoms)have been substituted with replacement atoms (or groups of atoms) havingsimilar steric and/or electronic features to those atoms which theyreplace. The substitution of a hydrogen atom or a hydroxyl group with afluorine atom is a commonly employed bioisosteric replacement.Sila-substitution (C/Si-exchange) is a relatively recent technique forproducing isosteres. This approach involves the replacement of one ormore specific carbon atoms in a compound with silicon (for a review, seeTacke and Zilch (1986) Endeavour, New Series 10: 191-197). Thesila-substituted isosteres (silicon isosteres) may exhibit improvedpharmacological properties, and may for example be better tolerated,have a longer half-life or exhibit increased potency (see for exampleEnglebienne (2005) Med. Chem., 1(3): 215-226). Similarly, replacement ofan atom by one of its isotopes, for example hydrogen by deuterium, mayalso lead to improved pharmacological properties, for example leading tolonger half-life (see for example Kushner et al (1999) Can J PhysiolPharmacol. 77(2):79-88). In its broadest aspect, the present inventioncontemplates all bioisosteres (and specifically, all siliconbioisosteres) of the compounds of the invention.

The terms derivative and pharmaceutically acceptable derivative asapplied to the compounds of the invention define compounds which areobtained (or obtainable) by chemical derivatization of the parentcompound of the invention. The pharmaceutically acceptable derivativesare therefore suitable for administration to or use in contact with thetissues of humans without undue toxicity, irritation or allergicresponse (i.e. commensurate with a reasonable benefit/risk ratio).Preferred derivatives are those obtained (or obtainable) by alkylation,esterification or acylation of the parent compounds. Thus, thepharmaceutically acceptable derivates of the compound of the inventionincludes N-oxides and esters thereof.

The pharmaceutically acceptable derivatives of the invention may retainsome or all of the biological activities described herein. In somecases, the biological activity is increased by derivatization. Thederivatives may act as pro-drugs, and one or more of the biologicalactivities described herein may arise only after in vivo processing.Particularly preferred pro-drugs are ester derivatives which areesterified at one or more of the free hydroxyls and which are activatedby hydrolysis in vivo. Derivatization may also augment other biologicalactivities of the compound, for example bioavailability and/orglycosidase inhibitory profile. For example, derivatization may increaseCNS penetration (e.g. penetration of the blood-brain barrier).

The term pharmaceutically acceptable salt defines any non-toxic organicor inorganic acid addition salt of the free base which are suitable foruse in contact with the tissues of humans and lower animals withoutundue toxicity, irritation, allergic response and which are commensuratewith a reasonable benefit/risk ratio. Suitable pharmaceuticallyacceptable salts are well known in the art. Examples are the salts withinorganic acids (for example hydrochloric, hydrobromic, sulphuric andphosphoric acids), organic carboxylic acids (for example acetic,propionic, glycolic, lactic, pyruvic, malonic, succinic, fumaric, malic,tartaric, citric, ascorbic, maleic, hydroxymaleic, dihydroxymaleic,benzoic, phenylacetic, 4-aminobenzoic, 4-hydroxybenzoic, anthranilic,cinnamic, salicylic, 2-phenoxybenzoic, 2-acetoxybenzoic and mandelicacid) and organic sulfonic acids (for example methanesulfonic acid andp-toluenesulfonic acid).

These salts and the free base compounds can exist in either a hydratedor a substantially anhydrous form. Crystalline forms, including allpolymorphic forms, of the iminosugars of the invention are alsocontemplated and in general the acid addition salts of the compounds arecrystalline materials which are soluble in water and various hydrophilicorganic solvents and which in comparison to their free base forms,demonstrate higher melting points and an increased solubility.

The term pharmaceutically acceptable metabolite as applied to thecompounds of the invention defines a pharmacologically active productproduced through metabolism in the body of the specified compound orsalt thereof.

The term pharmaceutically acceptable prodrug as applied to the compoundsof the invention defines any pharmaceutically acceptable compound thatmay be converted under physiological conditions or by solvolysis to thespecified compound, to a pharmaceutically acceptable salt of suchcompound or to a compound that shares at least some of the activity ofthe specified compound.

Prodrugs and active metabolites of the compounds of the invention may beidentified using routine techniques known in the art (see for example,Bertolini et al., J. Med. Chem., 1997, 40, 2011-2016).

In the present specification the term “alkyl” defines a straight orbranched saturated hydrocarbon chain. The term “C₁-C₆ alkyl” refers to astraight or branched saturated hydrocarbon chain having one to sixcarbon atoms. The term “C₁-C₉ alkyl” refers to a straight or branchedsaturated hydrocarbon chain having one to nine carbon atoms. The term“C₁-C₁₅ alkyl” refers to a straight or branched saturated hydrocarbonchain having one to fifteen carbon atoms. Preferred is C₁-C₆ alkyl.Examples include methyl, ethyl, n-propyl, isopropyl, t-butyl, n-hexyl.The alkyl groups of the invention may be optionally substituted by oneor more halogen atoms.

In the present specification the term “alkenyl” defines a straight orbranched hydrocarbon chain having containing at least one carbon-carbondouble bond. The term “C₁-C₆ alkenyl” refers to a straight or branchedunsaturated hydrocarbon chain having one to six carbon atoms. The term“C₁-C₉ alkenyl” refers to a straight or branched unsaturated hydrocarbonchain having one to nine carbon atoms. The term “C₁-C₁₅ alkenyl” refersto a straight or branched unsaturated hydrocarbon chain having one tofifteen carbon atoms. Preferred is C₁-C₆ alkenyl. Examples includeethenyl, 2-propenyl, and 3-hexenyl. The alkenyl groups of the inventionmay be optionally substituted by one or more halogen atoms.

In the present specification the term “alkynyl” defines a straight orbranched hydrocarbon chain having containing at least one carbon-carbontriple bond. The term “C₁-C₆ alkynyl” refers to a straight or branchedunsaturated hydrocarbon chain having one to six carbon atoms. The term“C₁-C₉ alkynyl” refers to a straight or branched unsaturated hydrocarbonchain having one to nine carbon atoms. The term “C₁-C₁₅ alkynyl” refersto a straight or branched unsaturated hydrocarbon chain having one tofifteen carbon atoms. Preferred is C₁-C₆ alkynyl. Examples includeethynyl, 2-propynyl, and 3-hexynyl. The alkynyl groups of the inventionmay be optionally substituted by one or more halogen atoms.

The term isolated as applied to the ido-BR1 of the invention is usedherein to indicate that the ido-BR1 exists in a physical milieu distinctfrom that in which it occurs in nature (or in the case of syntheticido-BR1, is purified to some degree). For example, the isolated ido-BR1may be substantially isolated (for example purified) with respect to thecomplex cellular milieu in which it naturally occurs (or with respect tothe some or all of the starting products, intermediates, buffers,solvents, reactants and/or co-products from which it is synthesised).

When the isolated material (e.g. synthetic, non-naturally occurringido-BR1) is purified, the absolute level of purity is not critical andthose skilled in the art can readily determine appropriate levels ofpurity according to the use to which the material is to be put.Preferred, however, are purity levels of 50% w/w, 60% w/w, 70% w/w, 80%w/w, 90% w/w, 99% w/w or higher. In some circumstances, the isolatedido-BR1 forms part of a composition (for example a more or less crudeextract containing many other substances) or buffer system, which mayfor example contain other components. In other circumstances, theisolated ido-BR1 may be purified to essential homogeneity, for exampleas determined spectrophotometrically, by NMR or by chromatography (forexample GC-MS of the trimethylsilyl-derivatives).

The term herbal medicine is used herein to define a pharmaceuticalcomposition in which at least one active principle (e.g. the ido-BR1) isnot chemically synthesized and is a phytochemical constituent of aplant. In most cases, this non-synthetic active principle is notisolated (as defined herein), but present together with otherphytochemicals with which it is associated in the source plant. In somecases, however, the plant-derived bioactive principle(s) may be in aconcentrated fraction or isolated (sometimes involving high degrees ofpurification). In many cases, however, the herbal medicine comprises amore or less crude extract, infusion or fraction of a plant or even anunprocessed whole plant (or part thereof), though in such cases theplant (or plant part) is usually at least dried and/or milled.

The term nutraceutical is used herein to define a food product (orisolate thereof) which provides physiological benefits or protectsagainst disease. Preferred nutraceuticals of the invention areanti-inflammatory.

The term standard specification is used herein to define acharacteristic, or a phytochemical profile, which is correlated with anacceptable quality of the herbal medicine, cosmetic or nutraceutical. Inthis context, the term quality is used to define the overall fitness ofthe product for its intended use, and includes the presence of ido-BR1at an appropriate concentration.

Posology

The compounds of the present invention can be administered topically orby oral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration.

The amount administered can vary widely according to the particulardosage unit employed, the period of treatment, the age and sex of thepatient treated, the nature and extent of the disorder treated, and theparticular compound selected.

Moreover, the compounds of the invention can be used in conjunction withother agents known to be useful in the treatment of diseases ordisorders arising from protein folding abnormalities (as describedinfra) and in such embodiments the dose may be adjusted accordingly.

In general, the effective amount of the compound administered willgenerally range from about 0.01 mg/kg to 500 mg/kg daily. A unit dosagemay contain from 0.05 to 500 mg of the compound, and can be taken one ormore times per day. The compound can be administered with apharmaceutical carrier using conventional dosage unit forms eitherorally, parenterally, or topically, as described below.

The preferred route of administration is oral administration. In generala suitable dose will be in the range of 0.01 to 500 mg per kilogram bodyweight of the recipient per day, preferably in the range of 0.1 to 50 mgper kilogram body weight per day and most preferably in the range 1 to 5mg per kilogram body weight per day.

The desired dose is preferably presented as a single dose for dailyadministration. However, two, three, four, five or six or more sub-dosesadministered at appropriate intervals throughout the day may also beemployed. These sub-doses may be administered in unit dosage forms, forexample, containing 0.001 to 100 mg, preferably 0.01 to 10 mg, and mostpreferably 0.5 to 1.0 mg of active ingredient per unit dosage form.

Formulation

The compound for use according to the invention may take any form. Itmay be synthetic or isolated from natural sources (for example from anyof the botanical sources identified herein, including for example abotanical source selected from plants of the family Cucurbitaceae (forexample, plants of the genus Cucumis, e.g. plants of the species Cucumissativus). Particularly preferred as botanical source are fruits of theforegoing plants, for example cucumbers, pumpkins, squashes or gourds.

When isolated from a natural source, the ido-BR1 may be purified.However, the compositions of the invention may take the form of herbalmedicines, as hereinbefore defined. Such herbal medicines preferably areanalysed to determine whether they meet a standard specification priorto use.

The herbal medicines for use according to the invention may be driedplant material. Alternatively, the herbal medicine may be processedplant material, the processing involving physical or chemicalpre-processing, for example powdering, grinding, freezing, evaporation,filtration, pressing, spray drying, extrusion, supercritical solventextraction and tincture production. In cases where the herbal medicineis administered or sold in the form of a whole plant (or part thereof),the plant material may be dried prior to use. Any convenient form ofdrying may be used, including freeze-drying, spray drying or air-drying.

Ido-BR1 may be separated from the higher molecular weight componentssuch as proteins and polysaccharides by using various membranetechnologies. These include microfiltration, ultrafiltration andnanofiltration. Alternatively, or in addition, electrodialysis may alsobe used to concentrate the charged ido-BR1. These methods use membranesof pore sizes that allow only molecules below a certain size to pass orrely on charges on the molecules to allow or not allow them to passthrough the membrane. Anion and cation exchange resins may also be usedto concentrate the ido-BR1.

When isolated from a natural source, the compound for use according tothe invention may be purified. In embodiments where the compound isformulated together with a pharmaceutically acceptable excipient, anysuitable excipient may be used, including for example inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavouring agents, colouring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc.

The pharmaceutical compositions may take any suitable form, and includefor example tablets, elixirs, capsules, solutions, suspensions, powders,granules and aerosols.

The pharmaceutical composition may take the form of a kit of parts,which kit may comprise the composition of the invention together withinstructions for use and/or a plurality of different components in unitdosage form.

Tablets for oral use may include the compound for use according to theinvention, mixed with pharmaceutically acceptable excipients, such asinert diluents, disintegrating agents, binding agents, lubricatingagents, sweetening agents, flavouring agents, colouring agents andpreservatives. Suitable inert diluents include sodium and calciumcarbonate, sodium and calcium phosphate, and lactose, while corn starchand alginic acid are suitable disintegrating agents. Binding agents mayinclude starch and gelatin, while the lubricating agent, if present,will generally be magnesium stearate, stearic acid or talc. If desired,the tablets may be coated with a material such as glyceryl monostearateor glyceryl distearate, to delay absorption in the gastrointestinaltract. Capsules for oral use include hard gelatin capsules in which thecompound for use according to the invention is mixed with a soliddiluent, and soft gelatin capsules wherein the active ingredient ismixed with water or an oil such as peanut oil, liquid paraffin or oliveoil.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate. Formulations suitable for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing in addition to the active ingredient suchcarriers as are known in the art to be appropriate.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the compounds of the invention will generally be provided in sterileaqueous solutions or suspensions, buffered to an appropriate pH andisotonicity. Suitable aqueous vehicles include Ringers solution andisotonic sodium chloride. Aqueous suspensions according to the inventionmay include suspending agents such as cellulose derivatives, sodiumalginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agentsuch as lecithin. Suitable preservatives for aqueous suspensions includeethyl and n-propyl p-hydroxybenzoate.

The compounds of the invention may also be presented as liposomeformulations.

For oral administration the compound can be formulated into solid orliquid preparations such as capsules, pills, tablets, troches, lozenges,melts, powders, granules, solutions, suspensions, dispersions oremulsions (which solutions, suspensions dispersions or emulsions may beaqueous or non-aqueous). The solid unit dosage forms can be a capsulewhich can be of the ordinary hard- or soft-shelled gelatin typecontaining, for example, surfactants, lubricants, and inert fillers suchas lactose, sucrose, calcium phosphate, and cornstarch.

In another embodiment, the compounds of the invention are tableted withconventional tablet bases such as lactose, sucrose, and cornstarch incombination with binders such as acacia, cornstarch, or gelatin,disintegrating agents intended to assist the break-up and dissolution ofthe tablet following administration such as potato starch, alginic acid,corn starch, and guar gum, lubricants intended to improve the flow oftablet granulations and to prevent the adhesion of tablet material tothe surfaces of the tablet dies and punches, for example, talc, stearicacid, or magnesium, calcium, or zinc stearate, dyes, colouring agents,and flavouring agents intended to enhance the aesthetic qualities of thetablets and make them more acceptable to the patient.

Suitable excipients for use in oral liquid dosage forms include diluentssuch as water and alcohols, for example, ethanol, benzyl alcohol, andthe polyethylene alcohols, either with or without the addition of apharmaceutically acceptably surfactant, suspending agent or emulsifyingagent.

The compounds of the invention may also be administered parenterally,that is, subcutaneously, intravenously, intramuscularly, orinterperitoneally.

In such embodiments, the compound is provided as injectable doses in aphysiologically acceptable diluent together with a pharmaceuticalcarrier (which can be a sterile liquid or mixture of liquids). Suitableliquids include water, saline, aqueous dextrose and related sugarsolutions, an alcohol (such as ethanol, isopropanol, or hexadecylalcohol), glycols (such as propylene glycol or polyethylene glycol),glycerol ketals (such as 2,2-dimethyl-1,3-dioxolane-4-methanol), ethers(such as poly(ethylene-glycol) 400), an oil, a fatty acid, a fatty acidester or glyceride, or an acetylated fatty acid glyceride with orwithout the addition of a pharmaceutically acceptable surfactant (suchas a soap or a detergent), suspending agent (such as pectin, carhomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose), or emulsifying agent and other pharmaceuticallyadjuvants. Suitable oils which can be used in the parenteralformulations of this invention are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineraloil. Suitable fatty acids include oleic acid, stearic acid, andisostearic acid. Suitable fatty acid esters are, for example, ethyloleate and isopropyl myristate.

Suitable soaps include fatty alkali metal, ammonium, and triethanolaminesalts and suitable detergents include cationic detergents, for example,dimethyl dialkyl ammonium halides, alkyl pyridinium halides, andalkylamines acetates; anionic detergents, for example, alkyl, aryl, andolefin sulphonates, alkyl, olefin, ether, and monoglyceride sulphates,and sulphosuccinates; nonionic detergents, for example, fatty amineoxides, fatty acid alkanolamides, and polyoxyethylenepolypropylenecopolymers; and amphoteric detergents, for example, alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium salts, aswell as mixtures.

The parenteral compositions of this invention will typically containfrom about 0.5 to about 25% by weight of the compound for use accordingto the invention in solution. Preservatives and buffers may also beused. In order to minimize or eliminate irritation at the site ofinjection, such compositions may contain a non-ionic surfactant having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5 to about15% by weight. The surfactant can be a single component having the aboveHLB or can be a mixture of two or more components having the desiredHLB. Illustrative of surfactants used in parenteral formulations are theclass of polyethylene sorbitan fatty acid esters, for example, sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol.

The compound for use according to the invention may also be administeredtopically, and when done so the carrier may suitably comprise asolution, ointment or gel base. The base, for example, may comprise oneor more of the following: petrolatum, lanolin, polyethylene glycols, beewax, mineral oil, diluents such as water and alcohol, and emulsifiersand stabilizers. Topical formulations may contain a concentration of thecompound from about 0.1 to about 10% w/v (weight per unit volume).

When used adjunctively, the compound for use according to the inventionmay be formulated for use with one or more other drug(s). Thus,adjunctive use may be reflected in a specific unit dosage designed to becompatible (or to synergize) with the other drug(s), or in formulationsin which the compound is admixed with one or more enzymes. Adjunctiveuses may also be reflected in the composition of the pharmaceutical kitsof the invention, in which the compounds of the invention is co-packaged(e.g. as part of an array of unit doses) with the enzymes. Adjunctiveuse may also be reflected in information and/or instructions relating tothe co-administration of the compound and/or enzyme.

Cosmetic Formulations

The cosmetic compositions of the invention may be selected for examplefrom moisturizing compositions, cleansing compositions, or anycomposition that may provide a benefit to the skin. The cosmeticcompositions of the invention may comprise cosmetically-acceptableexcipients or carriers, for example selected from those described below.

In one embodiment, the cosmetic composition is a cleansing composition.Suitable cleansing compositions are solid or semi-solid at roomtemperature. Examples of useful cleansing compositions include, but arenot limited to, fatty acid soaps, including glycerin soaps, syntheticdetergents and mixtures thereof. Solid cleansing compositions areextensively taught in Soap Technology for the 1990's, the contents ofwhich are incorporated herein by reference. It is desirable that thecleansing composition be flowable.

In one embodiment of the invention, the cleansing composition comprisesglycerin soap. Examples of glycerin soaps useful in the presentinvention include but are not limited to those disclosed in U.S. Pat.Nos. 4,405,492 and 4,879,063, the disclosures of which are herebyincorporated by reference.

Examples of suitable fatty acid soaps include soaps derived fromhydrocarbon chain lengths of from approximately 10 to 22 (includingcarboxyl carbon) and may be saturated or unsaturated. The soap may be,for example, the sodium salt, potassium salt, ammonium salt,triethanolammonium salt and mixtures thereof.

Suitable synthetic detergents include those known in the art for thedesired purpose. Examples of detergents useful for personal cleansinginclude the isethionates, sarcosinates, and glyceryl ether sulfonateswhich may be pure chain length variants or those derived from commercialoils such as coconut oil. Other suitable detergents include anionic acylsarcosinates, methyl acyl taurates, N-acyl glutamates, alkylsulphosuccinates, alkyl phosphate esters, ethoxylated alkyl phosphateesters, trideceth sulphates, protein condensates, mixtures ofethoxylated alkyl sulphates and alkyl amine oxides, betaines, sultainesand mixtures thereof. Included are the alkyl ether sulphates with 1 to12 ethoxy groups, especially ammonium and sodium lauryl ether sulphates.

The cosmetic composition may be a moisturizing composition.

Other optional components of the cosmetic compositions of the inventioninclude, but are not limited to, perfumes, fragrances, preservatives,colourants, dyes, anti-caking agents, and personal care ingredients,including, but are not limited to, skin and hair care ingredients.

Examples of suitable personal care ingredients useful in the presentinvention include but are not limited to safe and effective amounts of:humectants, sunscreen actives, skin soothers, anti-irritants,anti-inflammatories, emollients, conditioning agents, moisturizers,deodorants, anti-perspirants, artificial tanning agents, antimicrobialagents, anti-acne agents, anti-wrinkle agents, anti-skin atrophy agents,skin firming agents, anti-itch agents, anti-fungal agents, topicalanaesthetics, skin tone evening agents, active natural ingredients,agents for minimizing the appearance or retarding regrowth of unwantedhair, skin texture modifiers, and additional cleansing agents.

In one embodiment the ido-BR1 may be used from a water or alcoholicwater extract by using a water in oil (w/o) emulsion such as areemployed for example in the treatment of dry skin and emollientapplications

Emollients function by their ability to remain on the skin surface or inthe stratum corneum to act as lubricants, to reduce flaking, and toimprove the skin appearance. Typical emollients include fatty esters,fatty alcohols, mineral oil, polyether siloxane copolymers and the like.Examples of suitable emollients include, but are not limited to,polypropylene glycol (“PPG”)-15 stearyl ether, PPG-10 cetyl ether,steareth-10, oleth-8, PPG-4 lauryl ether, vitamin E acetate, PEG-7glyceryl cocoate, lanolin, and combinations thereof. Vitamin E acetate,PEG-7 glyceryl cocoate and combinations thereof are preferred.

Examples of suitable humectants include polyhydric alcohols. Suitablepolyhydric alcohols include, but are not limited to, glycerol (alsoknown as glycerin), polyalkylene glycols, alkylene polyols and theirderivatives, including propylene glycol, dipropylene glycol,polypropylene glycol, polyethylene glycol and derivatives thereof,sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-dibutyleneglycol, 1,2,6,-hexanetriol, ethoxylated glycerol, propoxylated glyceroland mixtures thereof.

Suitable skin soothers include, but are not limited to, panthenol,bisabolol, allantoin, aloe, and combinations thereof.

Suitable conditioning agents include, but are not limited to,dimethicone propyl PG-betaine, dimethicone copolyols, polyquaternium-10,guar, guar derivatives, and combinations thereof. Suitable anti-acneactive ingredients include, but are not limited to, salicylic acid,sulphur, lactic acid, glycolic acid, pyruvic acid, urea, resorcinol,N-acetylcysteine, retinoic acid, benzoyl peroxide, octopirox, triclosan,azelaic acid, phenoxyethanol, phenoxypropanol, flavinoids, derivativesthereof, and combinations thereof. Salicylic acid and benzoyl peroxideare preferred.

EXEMPLIFICATION

The invention will now be described with reference to specific Examples.These are merely exemplary and for illustrative purposes only: they arenot intended to be limiting in any way to the scope of the monopolyclaimed or to the invention described. These examples constitute thebest mode currently contemplated for practicing the invention.

Example 1 Identification of Ido-BR1 in Cucumber

Extraction of cucumber fruits in water or aqueous solvents such as 70%aq. ethanol is effective at extracting the ido-BR1. Cation exchangeresins such as IR120 or Dowex 50 resin in the H⁺ form (or similar formselected by those practiced in the art) will provide a concentratedfraction containing ido-BR1 along with amino acids. Use of an anionexchange resin such as CG400 or Dowex 1 in the OH⁻ form (or similar formselected by those practiced in the art) will also provide anido-BR1-enriched fraction.

GC-MS

All samples from ion exchange columns were freeze dried beforederivatization. Trimethylsilyl (TMS) derivatives were prepared using amixture of hexamethyldisilazane and trimethylchlorosilane in pyridine(Pierce Tri-Sir silylation reagent, HMDS:TMCS:pyridine in a ratio of2:1:10). Samples were heated at 60° C. for 15 minutes and then left atroom temperature for at least 60 min. Insoluble reaction products weresedimented by centrifugation, and the supernatant was transferred tofresh vials using a syringe.

Analysis was carried out by GC-MS using a Perkin Elmer Autosystem XL gaschromatograph with a high polarity fused-silica column (Varian ‘FactorFour’ VF-5 ms column, 25 m×0.25 mm i.d., 0.25 μm phase thickness). Thecarrier gas (helium) flow rate was 1 ml min-1. Trimethylsilyl—(TMS)derivatives were separated using a temperature programme that started at160° C. for 5 min, followed by a linear increase to 300° C. at a rate of10° C. min-1. Electron impact mass spectrometry of the column eluant wascarried out using a Perkin Elmer TurboMass Gold mass spectrometer, witha quadrupole ion filter system, which was run at 250° C. constantlyduring analysis. The injection volume was 1 μl.

Ido-BR1 gives a distinctive mass spectrum as thetrimethylsilyl-derivative with major fragments seen at 147, 217, 258,330 and 420 amu. It is clearly distinguishable in cucumber extract atconcentrations comparable to the primary amino acids such as asparticacid. Using the GC method described ido-BR1 has a retention time of10.45 minutes with the common amino acids seen at 3-5 minutes. Theretention time of ido-BR1 is comparable to that of thetrimethylsilyl-derivatives of glucose. The removal of glucose by the ionexchange methods described allows the determination of ido-BR1 by GC-MS.No other iminosugars or iminosugar acids are present in cucumber fruits.

Example 2 Confirmation of the Structure of Ido-BR1

Purification of the cucumber ido-BR1 is complicated by the common aminoacids that are present at similar concentrations in the fruit. However,after concentration using cation and anion exchange chromatography it ispossible to further purify the ido-BR1 by passing it down a neutralalumina oxide column and eluting with water to give a nearly purecompound (JH0808/155/water) that could be used for proton and carbon NMRto confirm the structure by comparison with synthetic ido-BR1.

Example 3 Glycosidase Assays

All enzymes and para-nitrophenyl substrates were purchased from Sigma,with the exception of beta-mannosidase which came from Megazyme. Enzymeswere assayed at 27° C. in 0.1 M citric acid/0.2M disodium hydrogenphosphate buffers at the optimum pH for the enzyme. The incubationmixture consisted of 10 μl enzyme solution, 10 μl of 1 mg/ml aqueoussolution of sample and 50 μl of the appropriate 5 mM para-nitrophenylsubstrate made up in buffer at the optimum pH for the enzyme. Thereactions were stopped by addition of 70 μl 0.4M glycine (pH 10.4)during the exponential phase of the reaction, which had been determinedat the beginning using uninhibited assays in which water replacedinhibitor. Final absorbances were read at 405 nm using a Versamaxmicroplate reader (Molecular Devices). Assays were carried out intriplicate, and the values given are means of the three replicates perassay. The method was carried out as described in Watson et al. (1997)Phytochemistry 46 (2): 255-259.

The table below shows the percentage inhibition caused by DNJ, BR1 andido-BR1 when tested on a panel of glycosidases at 0.2 mM. A negativevalue suggests a stabilisation of the enzyme or promotion of the enzymeactivity (perhaps by binding to a non-catalytic site). DNJ shows potentinhibition of glucosidases but BR1 is a poor inhibitor and ido-BR1 veryweakly inhibitory to the two glucosidases it was tested on. BR1 is agood inhibitor of glucuronidase but ido-BR1 is not inhibitory. Ido-BR1appears to promote alpha-mannosidase activity slightly. From theseresults we suggest that ido-BR1 will not inhibit digestive glucosidasessignificantly and therefore will have a better drug profile than DNJ forinflammatory disorders.

6-epi- Assay DNJ BR1 BR1 α-D-glucosidase 35.7 4.9 4.7 (Yeast)α-D-glucosidase 99.5 49.2 26.4 (Bacillus) α-D-glucosidase 99.6 55.2 —(Rice) β-D-glucosidase 63.7 6.7 6.1 α-D-galactosidase −3.3 −6.3 −2.7β-D-galactosidase 0.4 6.2 1.0 α-L-fucosidase 5.9 1.6 — α-D-mannosidase26.6 28.9 −28.4 β-D-mannosidase −19.0 7.8 −4.4 Naringinase 20.8 1.4 —N-acetyl-β-D-gluc −0.8 2.4 5.5 (Bovine kidney) N-acetyl-β-D-gluc 2.810.4 −4.5 (Jack bean) N-acetyl-β-D- −5.5 −1.1 — hexosaminidaseAmyloglucosidase 31.7 5.5 — β-glucuronidase — 88.9 2.5

Example 4 Ido-BR1 Abrogates Inflammation 24 h After Challenge in an InVitro Model

The efficacy of ido-BR1 as an anti-inflammatory agent in a model ofmucosal inflammation was tested. The model utilised was previouslyoptimised and established (D M Nash, E A. Lane, S Herath and I M Sheldon(2008) Endometrial Explant Culture for Characterizing EquineEndometritis. American Journal of Reproductive Immunology. 59: 105-117).The model is comprised of an in vitro equine endometrial explant tissueculture system. There are several advantages of using uterine tissuecollected from horses: large uterine surface area to harvest copioustissue enabling several treatments and replicates in a single experiment(ensures statistical robustness); directly representative of wholeanimal clinical inflammation in the horse (uterine inflammation is asignificant cause of subfertility), and indirectly of other species(e.g. cattle and humans) and inflammation at other mucosal surfaces(e.g. gut and lung).

Uterine tissue was collected from mares at an abattoir, the endometrial(mucosal) surface was dissected out, homogenised and placed in cultureas explants. In order to stimulate inflammation synonymous to that whichoccurs with natural uterine infection in the whole animal, the O-antigenof E. coli, lipopolysaccharide (LPS), was added to the culturedexplants. Inflammation was indicated by measuring the secretion of anestablished marker of inflammation, Prostaglandin F_(2α) (PGF_(2α)), byexplants from the culture media. The secretion of PGF_(2α) is measuredat 24 and 72 h after challenge with LPS in order to represent the innateand chronic immune response respectively.

Explants were treated with control (media alone) or, in order todetermine whether ido-BR1 affected spontaneous PGF_(2α), 0.5, 25 or 100μg/ml ido-BR1 alone. In order to test the ability of ido-BR1 to abrogatethe inflammatory response, explants were treated with 3.0 μg/ml LPSalone, or 3.0 μg/ml LPS in the presence of 0.5, 25 or 100 μg/ml ido-BR1.The secretion of PGF_(2α) was measured at 24 and 72 h after treatmentswere applied. Explants were collected from 4 different animals (n=4) andeach treatment was conducted using explants cultured in triplicate foreach animal.

Twenty four hours after treatment, explants that were exposed to theido-BR1 alone (without LPS), secreted PGF_(2α) concentrations that weresimilar (not significantly different) to the control. Therefore, theido-BR1 itself did not inadvertently stimulate inflammation in theexplants. Explants treated with LPS alone secreted PGF_(2α)concentrations that were significantly greater than that of the control,indicating inflammation was stimulated by LPS. Explants that weretreated with LPS and ido-BR1 concurrently secreted PGF_(2α)concentrations that were not significantly different to the control.

Therefore, the ido-BR1 abrogated inflammation in the explants. Theabrogation of inflammation by ido-BR1 occurred in a dose-dependentmanor. However, the effect, although showing a similar pattern, was notsignificant at 72 h.

EQUIVALENTS

The foregoing description details presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare intended to be encompassed within the claims appended hereto.

The invention claimed is:
 1. A cosmetic, nutraceutical, herbal medicineor pharmaceutical composition comprising(2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid orpharmaceutically acceptable salt thereof, isolated from a botanicalsource comprising plants of the genus Cucumis, and a cosmetically-,nutraceutically- or pharmaceutically-acceptable excipient or carrier. 2.A cosmetic method for the reduction of swelling or erythema of the skincomprising administration of a composition comprising(2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid orpharmaceutically acceptable salt thereof, isolated from a botanicalsource, to a subject in need thereof.
 3. A method for treating aninflammatory disease or for reducing inflammation comprisingadministering an effective amount of a composition comprising(2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid orpharmaceutically acceptable salt thereof, isolated from a botanicalsource, to a subject in need thereof.
 4. A process for the production ofa cosmetic, nutraceutical or pharmaceutical composition comprising(2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid, orpharmaceutically acceptable salt thereof, and a cosmetically-,nutraceutically- or pharmaceutically-acceptable excipient or carrier,said process comprising the steps of: (a) providing plant material froma botanical source comprising plants of the genus Cucumis; (b)extracting (2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid orpharmaceutically acceptable salt thereof from said plant material; and(c) formulating said(2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid orpharmaceutically acceptable salt thereof with a cosmetically-,nutraceutically- or pharmaceutically-acceptable excipient or carrier toproduce the cosmetic, nutraceutical or pharmaceutical composition.
 5. Acosmetic, nutraceutical or pharmaceutical composition obtainable by theprocess of claim
 4. 6. The composition of claim 1, wherein the(2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid orpharmaceutically acceptable salt is present in the composition at alevel of up to 1% w/w (dry weight basis).
 7. The composition of claim 1,wherein the botanical source comprises plants of the species Cucumissativus.
 8. The composition of claim 1, wherein the botanical source arecucumber fruits.
 9. The method of claim 2, wherein the administration isby topical application to the skin.
 10. The method of claim 2, whereinthe botanical source comprises plants of the genus Cucumis.
 11. Themethod of claim 2, wherein the botanical source comprises plants of thespecies Cucumis sativus.
 12. The method of claim 2, wherein thebotanical source are cucumber fruits.
 13. The method of claim 3, whereinthe botanical source comprises plants of the genus Cucumis.
 14. Themethod of claim 3, wherein the botanical source comprises plants of thespecies Cucumis sativus.
 15. The method of claim 3, wherein thebotanical source are cucumber fruits.
 16. The process of claim 4,wherein the botanical source comprises plants of the species Cucumissativus.
 17. The process of claim 4, wherein the botanical source arecucumber fruits.
 18. The process of claim 4, wherein the extracted(2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid orpharmaceutically acceptable salt thereof is present in the compositionat a level of at least 1% w/w (on a dry weight basis).
 19. The processof claim 4, wherein the extracted(2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid orpharmaceutically acceptable salt thereof is present in the compositionat a level of up to 1% w/w (dry weight basis).
 20. The process of claim4, further comprising detecting the presence or absence or measuring theamount of (2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid orpharmaceutically acceptable salt thereof present in a sample of saidcosmetic, nutraceutical or pharmaceutical composition.
 21. The processof claim 4, further comprising adding the cosmetic, nutraceutical orpharmaceutical composition to a foodstuff or beverage to provide asupplemented foodstuff or beverage.